User:Fraserhof/InVivoBioPrinting

In vivo bioprinting is the printing of tissues and/or organs directly on the site in need of reparation or regeneration. The body is used as a bioreactor. In vivo bioprinting is an extension of three dimensional bioprinting, the more general form of bioprinting. It has many advantages including precision and purity. However, there are some limitations such as biomaterials, time and vascularity.

The technologies used for in vivo bioprinting include droplet-based, extrusion-based and laser-based bioprinting. The approaches used are robotic arms or handheld devices, both of which are minimally invasive.

Currently, in vivo bioprinting has been used for the regeneration of skin, cartilage, tissues and bones. The reconstruction of organs has not yet been done and there have been no in vivo bioprinters used in clinical settings thus far. However, the goal for this technique is to be used on humans.

General applications
In vivo bioprinting has been widely used in tissue engineering because of its high resolution and precision. Due to the insufficient vascularization of tissue, it is hard to fabricate human-scale tissue. Because of this, in vivo bioprinting is only used to construct small tissues or organs at clinical relevance at this point in time. There are 3 major application of in vivo bioprinting: Bone, skin, and cartilage tissues.

Bone tissue
The most common researched area in tissue engineering is bone tissue. Bone tissue injuries and defects happen easily in the human body. Customary clinical operations use allografts or xenografts to repair bone tissue, but the risk of infection and rejection of the organ is relatively high.

In vivo bioprinting can avoid these problems with the main Laser-assisted bioprinting method. Two main pieces of research show in vivo 3D bone tissue printing for calvaria reparation in mice. Use of Laser-assisted bioprinting to print mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite motivate bone regeneration in mice. After 3 months, the immunodeficient mice had fully recovered with mature bone tissue. Another study uses Laser-assisted bioprinting to print a cell‐seeded biomaterial layer onto an electrospun polycaprolactone (PCL) scaffold. This results in a thicker fibrous tissue formed on the calvarial defect.

Skin tissue
Skin tissue contains many different cell types and functional structures. Although there are some commercial skin substituted products for skin problems, it is still hard to satisfy personalized skin problems.

In vivo bioprinting has a broad usage for inner-body repair. A thermal inkjet bioprinter is used to print amniotic fluid‐derived stem (AFS) cells onto a hydrogel, maintaining vasculature. The hydrogel is then implanted onto the defective site. A study showed that wound closure for in vivo procedures are quicker than in traditional surgery. Printing hydrogel structures demonstrates the capacity of the device for in vivo fabrication of 3D tissue scaffolds as well as the application prospect in the clinical field. The Laser-assisted bioprinting technique is also used to arrange cells according to the designed 3D spatial pattern.

Cartilage tissue
Cartilage repair and replacement is a major challenge in plastic reconstructive surgery since cartilage cannot be sensed or self-repaired in the body. Normally, during cartilage repair infections from multiple surgeries arise. This same problem happens when repairing bone tissues. To directly repair the cartilage tissue with 3D constructs, in vivo bioprinting is used.

Once again, similar to bone tissue bioprinting, nano-fibrillated cellulose, alginate, human chondrocytes and human mesenchymal stem cells are printed onto a hydrogel scaffold with a 3D-extrusion bioprinter. After the construct is implanted in the body, the production of chondrocyte and type 2 collagen can be observed. This demonstrates new in vivo cartilage growth.

Methods for in vivo bioprinting
Generally, the methods used in vivo are similar to those used in three dimensional bioprinting. The main difference is how these methods are utilized in an in vivo setting. Before the printing is conducted, the 3D structure of the biological object needs to be determined. The 3D structure can be determined in many ways using computed tomography (CT), magnetic resonance imaging (MRI), 3D scanners, and ultrasound imaging. Once the 3D construct is determined, this being through the use of computer-aided design (CAD) software, printing of the biological substrate can proceed. Although most of these methods are utilized in vitro, there is a trend moving towards in vivo applications. Recently, patents for in vivo bioprinting technologies have been filed demonstrating the viability of such methods.

Ink-jet based
Some of the first in vivo bioprinting techniques performed were based on inkjet technology. Now that three dimensional bioprinting has become more common-practice, development behind the methods and technology has made significant progress. Main advantages behind this style of printing is the low-cost nature, ease of use, and wide selection of printers.

Ink-jet based bioprinters include both thermal and piezoelectric drop-on-demand methods of bioprinting. A thermal bioprinter has a heating element rapidly increases the temperature of the bioink. In turn the rapid increase of temperature creates a bubble and the bioink is then extruded out of the orifice. Another model is the piezoelectric method. Crystals are located opposite to the nozzle and the crystals are subjected to an electric charge. Piezoelectric crystals vibrate under an electrical current, causing the bioink to be dispensed.

Although there has been progress behind the development of ink-jet based bioprinters there are still major limitations. One of these being the low viability of biomaterials being extruded through a small orifice. Another limitation is their lack of usability outside of repair and fabrication of superficial tissues. This issue comes down to two parts, the current bulky size of the printers as well as the lack of effective structural integrity.

Laser based
Laser induced forward transfer (LIFT) is the main basis behind laser assisted bioprinting (LAB). Main components of LAB are a pulsed laser beam, a substrate for deposition, deposition material (bioink, cells, hydrogel), a focussing system, and a ribbon. This ribbon is made up of glass which is covered in a laser energy absorbing layer, this being a metal such as titanium or gold.

Although not as common as ink-jet based bioprinting, LAB has some distinct advantages, including disadvantages. Due to the lack of nozzle, clogging is not seen as an issue as it is with ink-jet based bioprinting. On top of this, LAB systems can accommodate a range of viscosities and cell viability is negligible for mammalian cells. Some of the main concerns behind LAB are metallic contamination and its high cost of use. The metallic contamination in the printed biological object comes from the ribbons used. Due to this downfall work is being put into finding a ribbon substrate that will not affect printed materials.

As a proof of concept LAB was used to treat calvarial bone defects in mice using nano-hydroxyapatite.

Extrusion based
Extrusion based bioprinting is deposited by two main methods, pneumatic or mechanical. This method is most similar to non-biological 3D printing. Due to the simplistic nature of this printing method it has been developed for handheld in situ bioprinting which can directly translate to in vivo bioprinting.

Because of the simple design and history behind it, there are some clear benefits. Extrusion based printing’s origin, being non-biological 3D printing, allows it to print a wide range of viscosities. The main benefit behind this is improved structural integrity. Furthermore, the simple design allows for use in a variety of environments and provides an option for scalability which is needed for in vivo bioprinting.

Bio-electrospray and Cell Electrospinning
Electrospray and electrospinning techniques use an electric field to dispense media as droplets or fibers. This method has not been well studied for its use in vivo although it is similar to both laser and ink-jet based methods. The main concern with this method is the high voltages used during the bioprinting process.

Materials used in printing
The main material utilized while bioprinting is bioink. This material is built up of many more biologically compatible materials in order to fulfill the requirements behind bioprinting.

Many factors need to be considered while sourcing the contents of bioink used. One main factor is its biocompatibility. In order for the printed construct to be accepted by the organism it must not elicit an inflammatory or immune response from the host. Some hydrogels used include but are not limited to, naturally occurring polymers, or synthetic molecules. Naturally occurring polymers may be collagen, gelatin, or even fibrin, these are usually isolated from animal tissues. An example of a synthetic molecule is polyethylene glycol.

Another requirement behind the development of printable materials is printability and mechanical properties. Depending on required mechanical properties for the site of printing the viscosity needs to be closely monitored. One method of ensuring structural rigidity while using a low viscosity material is crosslinking although this comes with certain limitations.

Cell aggregates are the main ingredient for bioinks with in vivo bioprinting. This material would need to be combined with some hydrogel in order to increase the structural rigidity. The benefits behind cell aggregates is their self-organization and assembly which eventually form the tissue. A main drawback of cell aggregates is rejection by the host immune system. A workaround for this is obtaining a biopsy from the patient to match cell types.

Comparisons between in vitro and in vivo
There are many advantages of in vivo bioprinting over in vitro bioprinting. The main benefit is that in vivo bioprinting can print tissues or organs directly onto the damaged site. Using the body as the bioreactor, potential impurities are minimized. In comparison, in vitro bioprinting needs an artificial microenvironment. This is necessary for maturation of the new tissues and organs but causes several consequences. Once the fragile tissue or organ has been constructed, contamination, swelling or damage may occur during transportation. In addition, the physical properties of the newly designed tissue or organ may not be the correct size. With in vivo bioprinting, since the organ or tissue is being constructed directly at the defective location, there is higher purity and precision. In addition, there is a longer operating time for in vitro bioprinting due to doubtless adjustments.

The methods for in vivo and in vitro bioprinting also differ. The two techniques used for in vivo reconstruction of tissues and organs are robotic arms and handheld devices. Both are minimally invasive and have better access to the defective location. In comparison, the most common method used for in vitro bioprinting is a nozzle where the bioink is activated through temperature or electricity. This approach can be invasive and is not as suitable for in vivo bioconstruction as other methods.

Another difference between in vitro and in vivo bioprinting is the cost. In vivo bioprinting is more expensive. This is due to complex reparation or construction of organs and tissues. In vivo bioprinting can also produce heterogenous tissues on complicated surfaces while in vitro bioconstruction produces homogenous tissues on flat surfaces. To contrast, in vivo bioprinting is usually one surgery whereas in vitro bioprinting may need numerous procedures. Safety, shipping and shelf-life will also add to in vitro cost.

In vitro bioprinting is realistic for short-term application whereas in vivo bioprinting is good for long-term use. Furthermore, in vitro applications can be studied for future purposes, whereas in vivo bioprinting will be used in medical procedures.

Limitations
Although in vivo bioprinting has been found to be advantageous, there are some limitations.

Firstly, choosing the right biomaterial is important. Conventional 3D bioprinting material is not compatible for the in vivo procedure. It is biologically active and will therefore cause early and/or unwanted stem cell differentiation. A possible solution is to use a mixture of different biomaterials. For in vivo bioprinting, a firm scaffold for structure and a soft substance for tissue reproduction is needed. The correct biomaterial will guarantee good integration and development as well as reduce the chance of rejection.

Another challenge present is tissues printed with in vitro 3D bioprinters need external environmental cues throughout growth to generate phase changes. These signals include, UV light curing, chemical exposure, and temperature change. These external cues also cause the solidification of the bioink. Without the signals, an instantly solidifying bioink is essential. However, the cues are unsuited for in vivo surgery since they require invasive surgery and can negatively impact healthy tissues. Furthermore, in vivo bioprinting requires numerous cells. To guarantee sufficient numbers, in vitro bioprinting is used in conjunction with in vivo techniques, increasing the overall procedure time.

Moreover, vasculature is significant for in vivo bioprinting. It transports blood throughout the body, prevents tissue death and maintains homeostatic functions. Vasculature is more extensive on the surface of the body but is insufficient deeper within the body. The vascular network ensures sufficient nutrients and oxygen reach tissues (The effect of PEGT/PBT scaffold architecture on oxygen gradients in tissue engineered cartilaginous constructs). This is especially important for nutrients and oxygen to reach the newly developed organs and tissues. However, the rarity of vasculature deep within the body creates a problem for in vivo bioconstruction.

Another challenge is the printing speed needed for in vivo bioprinting. Time is vital during a clinical procedure. The length of the surgery will depend on the size, shape and function of the organ or tissue needed. If the speed of the bioprinting is too slow, it could be detrimental for the patient.

Additionally, in vivo bioprinting will have a high cost. Although it decreases manufacturing price and amount of labour needed, the length of the surgery and therefore the price will rise. Unfortunately, this will restrict the patients able to afford in vivo bioprinting. This is a major drawback as those who most need this surgery may be unable to afford it. Furthermore, in vivo bioprinting raises ethical concerns. The scans needed will provide practitioners with medical information that the patient may be unaware of. This is a confidentiality issue. There is also a question of ownership. Is the organ and/or tissue the property of the clinician or the patient? This causes the eligibility for a patent to become more controversial. A patent is unable to be done on a “product of nature." In this case, it is hard to tell if the in vivo bioprinted organ or tissue is a human organism since it contains cells that are genetically identical to the real organ or tissue.

Future applications
In vivo bioprinting has many limitations. The application is still experimental and has not been used on the human body in a clinical setting. Currently, organ reconstruction and in vivo bioprinters. However, the high‐speed, precision and consistent reproducibility make in vivo bioprinting a promising treatment.

The future application for in vivo bioprinting will have a wider selective of biomaterial to form more complex tissues with additional components or cellular types in order to favour tissue regeneration. For example, in vivo bioprinting will be used as a rapidly deep injured skin tissue treatment whether they are in civilian hospitals or military field hospitals.

Different types of in vivo bioprinters will be available. As a technique allows printing without direct contact, fully automatic robotic printers can be directed by the surgeon to achieve precise cellular implantation at the micron to millimeter-scale in a sterile environment inside the operating room. The bioprinting device will design more operable and user-friendly, thus Surgeons do not have to work with an engineering complicated device. Cooperating with AI will make the bioprinting process smarter and precise. The choice between a robotic arm and a handheld operated device will depend on the anatomical location and complexity of the tissue or organ being fabricated.

Fabricating a living organ is the likely future application of in vivo bioprinting. The global organ shortage crisis have existed for long time. In vivo bioprinting an organ by using minimally invasive surgery is less painful, has shorter recovery time, and higher quality results. This makes it possible to repair important organ in the human body such as human heart, more convenient.